WO2019200764A1

WO2019200764A1 - Improved cellulose and preparation method and application therefor

Info

Publication number: WO2019200764A1
Application number: PCT/CN2018/095903
Authority: WO
Inventors: 阮栋梁; 王晖; 程发良
Original assignee: 东莞理工学院
Priority date: 2018-04-18
Filing date: 2018-07-17
Publication date: 2019-10-24
Also published as: US20200308310A1

Abstract

Improved cellulose and a preparation method and an application therefor. The structural formula of the improved cellulose is shown in formula (I), formula (II), or formula (III): wherein n is 2-7; the improved cellulose is obtained by means of modifying and substituting the cellulose. The cellulose is substituted by long chain compounds having terminal amino groups, such that the modified cellulose has long chain space and terminal amino groups, and can be used as a matrix for solid phase polypeptide synthesis (SPOT), increasing the reaction activity and reaction space, reducing the difficulty of polypeptide synthesis, better implementing SPOT solid phase polypeptide synthesis, being suitable for synthesis by an automatic synthesiser, and having a high yield and low costs.

Description

[Name of invention established by ISA according to Rule 37.2] Modified cellulose and preparation method and application thereof

Technical field

The invention belongs to the field of polypeptide synthesis substrates, and particularly relates to a novel modified cellulose and a preparation method and application thereof.

Background technique

A polypeptide is a biologically active substance that is involved in various cellular functions in an organism. It is a kind of compound whose molecular structure is between amino acid and protein, and is composed of a plurality of amino acids combined by peptide bonds in a certain order. Now there is an unprecedented prosperity for the research and utilization of peptides. The chemical synthesis technology of peptides has matured both in liquid phase and solid phase. The structure of a new polypeptide can be verified by total peptide synthesis; a new polypeptide can be designed to study the relationship between structure and function, provide important information for the mechanism of polypeptide biosynthesis, establish model enzymes and synthesize new peptide drugs. The total synthesis of peptides not only has important theoretical significance, but also has important application value.

Through continuous improvement and improvement, the solid phase method has become a common technique in the synthesis of peptides and proteins, showing the advantages unmatched by classical liquid phase synthesis. For example, solid phase synthesis of peptides is a common method for peptide synthesis with its advantages of time saving, labor saving, material saving, computer control, and popularization. The basic principle of solid phase synthesis of peptide synthesis is a process of repeating the addition of amino acids. The synthesis is synthesized from the C-terminal (carboxy terminal) to the N-terminal (amino terminal): the hydroxyl group of the hydroxyl terminal amino acid of the desired peptide chain is covalently bonded. The structure is linked to an insoluble polymer resin, and then the amino acid bound to the solid phase carrier is used as an amino component to deprotect the amino group and react with an excess of the activated carboxyl component to lengthen the peptide chain. Repeat (condensation → washing → deprotection → neutralization and washing → next round condensation) operation to achieve the length of the peptide chain to be synthesized, and finally the peptide chain is cleaved from the resin, and after purification and the like, the desired polypeptide is obtained. The α-amino group is protected by BOC (tert-butoxycarbonyl), which is called BOC solid phase synthesis, and the α-amino group is protected by FMOC (9-fluorenylmethoxycarbonyl), which is called FMOC solid phase synthesis.

However, to synthesize a large number of peptides, such as the use of polypeptide chains for chemical screening to identify multi-ligand protein affinity reagents, standard solid phase peptide synthesis technology based on polymer resins appears to be slow and expensive; because the quality of the polypeptide required for chemical screening is small But the number is huge. In recent years, solid phase peptide synthesis has been developed on the principle of conventional resins (such as Wang resin and Rink resin), and several different modified substrates have been developed for peptide synthesis. These include tea-tea, digital lithography, pin synthesis and SPOT synthesis on cellulose. These improved techniques use standard peptide synthesis chemicals and building blocks in a very efficient manner and avoid peptide purification and analysis. The cost of SPOT polypeptide synthesis is dramatically reduced compared to standard solid phase peptide synthesis. Among them, the SPOT synthesis on cellulose is easy to operate, and can be performed manually, or a semi-automatic or fully automatic robot can be used. The number and quality of the peptide can be freely modified, and the detection operation of the peptide-ligand interaction is simple. The SPOT method follows standard Fmoc chemistry based on solid phase peptide synthesis on cellulose filters. The use of cellulose itself has several advantages over other materials: it is inexpensive and can tolerate the organic solvents and acids used in peptide synthesis. In addition, cellulose is stable in aqueous solution because it is non-toxic and is therefore suitable for screening biological samples, although the optimal range is between 6 and 18 amino acids.

Because cellulose is a polysaccharide composed of hundreds to tens of thousands of β-(1→4)-linked D-glucose units, its sugar hydroxyl group (-OH) and short-chain space lead to a solid phase directly as a matrix. The polypeptide synthesis conditions are stringent and the yield is low.

Therefore, the development of a modified cellulose as a matrix for solid-state peptide synthesis reaction makes the solid phase peptide synthesis reaction yield high and easy to carry out has important research significance and popularization value.

Summary of the invention

The object of the present invention is to overcome the defects of the prior art as a substrate for the synthesis reaction of solid polypeptides, which has strict conditions and low yield, and provides a novel modified cellulose. The improved cellulose provided by the invention replaces the sugar hydroxyl group on the cellulose by the long chain with amino group at the end, overcomes the problems caused by the sugar hydroxyl group and the short chain space in the cellulose, can reduce the synthesis condition of the SPOT solid phase polypeptide, and improves the production. Rate, greatly reducing the cost of solid phase peptide synthesis.

Another object of the present invention is to provide a process for the preparation of the above modified cellulose.

Another object of the present invention is to provide the use of the above improved cellulose as a matrix for the synthesis of solid polypeptides.

In order to achieve the above object, the present invention adopts the following technical solutions:

A novel modified cellulose, the structural formula of the modified cellulose is as shown in formula (I), formula (II) or formula (III):

Wherein n is 2-7; the modified cellulose is modified by cellulose substitution

The inventors of the present invention attempted to increase the short-chain space of cellulose by a long-chain compound to improve the performance of cellulose, and the selection of the functional group and chain length of the long-chain compound is a key factor. After many studies, it was found that when both ends of the functional group are amino groups, one amino terminal replaces the sugar hydroxyl group of cellulose to realize long-chain linkage; the other amino group provides hydroxyl synthesis of amino acids at the long-chain end. The space, while also achieving the same reaction conditions as the synthesis of the polypeptide, makes the automatic peptide synthesis easier; on the other hand, when the simplicity and repeatability of the repeating structure of the long-chain compound are selected, the performance is also very important. The length is too short (such as n = 1) to limit the length of the polypeptide; if the chain length is too long (such as (n > 7), the polypeptide synthesis yield is low.

Preferably, the structural formula of the modified cellulose is as shown in formula (II).

On the basis of the long-chain compound substitution modification, the Rink Amide linker is further modified to synthesize the solid phase polypeptide synthesized by using the modified cellulose as a matrix, and can be used as a bioactive peptide screening without cleavage; After scanning, the synthesized polypeptide can be cleaved from the Rink Amide linker matrix and further characterized by mass spectrometry or quantitative analysis.

Therefore, the modified cellulose modified by the Rink Amide linker provided by the present invention is subjected to Rink Amide linker modification by replacing the cellulose with a long-chain compound having a terminal amino group, so that the cellulose has a long chain space and a better reactivity. As a matrix, it is better to realize manual and automatic solid-state peptide synthesis with high yield and easy conditions.

Preferably, the structural formula of the modified cellulose is as shown in formula (III).

On the basis of the long-chain compound substitution modification, further modification is performed by Wang linker, and the modified cellulose is used as a matrix solid phase polypeptide synthesis, and the non-cleavage can be used as a bioactive peptide screening; The synthesized polypeptide can be cleaved from the matrix of Wang linker and further characterized by mass spectrometry or quantitative analysis.

Therefore, the Wang linker modified modified cellulose provided by the present invention replaces cellulose by a long-chain compound having a terminal amino group and then undergoes Wang linker modification, thereby making cellulose have a long chain space and a better solid phase polypeptide synthesis reaction activity. It can meet both manual and automatic synthesis of instruments. It can be used as a matrix to achieve manual and automatic solid-state peptide synthesis with high yield and easy conditions. At the same time, the peptide can be scanned for activity without cleavage after synthesis, and qualitative and quantitative analysis can be provided after cleavage.

Preferably, the cellulose is paper cellulose.

More preferably, the cellulose is a filter paper.

Preferably, n is 3.

The present invention provides a method for preparing the above modified cellulose, the method comprising the steps of:

S1: mixing a solution of cellulose and p-toluenesulfonyl chloride in pyridine, shaking, washing and air drying;

S2: adding long chain compounds

After the vibration is completed, the reaction is washed, and air-dried to obtain the modified cellulose of the formula (I);

S3: adding the Ring Amide resin and the activator to the modified cellulose of the formula (I), and then washing the modified cellulose to obtain the modified cellulose of the formula (II);

S4: adding the Wang linker and the activator to the modified cellulose of the formula (I), followed by washing and then washing to obtain the modified cellulose of the formula (III).

Preferably, the mass concentration of p-toluenesulfonyl chloride in the pyridine solution of the p-toluenesulfonyl chloride described in S1 is from 350 to 400 g/L.

More preferably, the mass concentration of tosyl chloride in the pyridine solution of p-toluenesulfonyl chloride described in S1 is 380 g/L.

Preferably, the activator in S3 is HOBt hydroxybenzotriazole and DIC N,N'-diisopropylcarbimine, or HOSu N-hydroxysuccinimide and DIC N,N'-diiso Propylcarbimine; the temperature of the reaction is 50 to 80 ° C, and the reaction time is 10 to 40 min.

More preferably, the temperature of the reaction is 70 ° C and the reaction time is 15 min.

Preferably, the activator in S4 is HMPA hexamethylphosphoric triamide and EEDQ 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; the temperature of the reaction is 18~ The reaction time is 12 to 16 hours at 25 °C.

More preferably, the temperature of the reaction is 20 ° C and the reaction time is 14 h.

Preferably, the step of treating the cellulose with water before the step S1 is further included.

Preferably, the solvent washed in S2 is dimethylformamide and dichloromethane.

The use of the above modified cellulose as a substrate in the synthesis of solid polypeptides is also within the scope of the present invention.

Compared with the prior art, the present invention has the following beneficial effects:

The invention replaces and modifys cellulose by a long-chain compound having a terminal amino group, so that the modified cellulose has a long chain space and a terminal amino group, and can be used as a matrix for solid polypeptide synthesis, thereby increasing reaction activity and reaction space. It is difficult to reduce the synthesis of the polypeptide, and the solid polypeptide synthesis is better, the yield is high, and the cost is low. In addition, the modified cellulose modified by Rink Amide linker is used as a matrix for solid phase peptide synthesis, and the bioactive peptide screening can be performed without cleavage; at the same time, after the activity scan, the synthesized polypeptide can be obtained from the Rink Amide linker substrate. After cutting, further synthesis or identification by mass spectrometry, or quantitative analysis; the modified cellulose further modified by Wang linker has long chain space and solid phase synthesis and reaction activity, which can simultaneously satisfy manual and instrumental automatic synthesis; The matrix is better for realizing solid peptide synthesis, and the yield is high and the conditions are easy; the non-cleavage can be used as a bioactive peptide screening, and the qualitative and quantitative analysis can be provided after the cutting.

DRAWINGS

Figure 1 is a sugar structure of paper cellulose;

2 is a schematic view showing a modified paper cellulose provided in Example 1 and a preparation process thereof;

Figure 3 is a schematic diagram of functional improvement of paper cellulose and synthesis of polypeptide;

Figure 4 is a (A) synthesized polypeptide sequence, (B) Intavis polypeptide synthesizer and modified paper cellulose-based synthetic polypeptide in UV light absorption and (C) Western Blotting results

5 is a schematic diagram of a modified cellulose modified by Rink Amide linker provided in Example 4 and a preparation process thereof;

Figure 6 is a graph showing the results of detecting a synthetic polypeptide (valine-valine-valine-valine-lysine) by liquid chromatography-mass spectrometry;

Figure 7 is a schematic view showing the steps of synthesizing a solid phase polypeptide;

Fig. 8 is a graph showing the results of detection of a synthetic polypeptide (valine-valine-valine-valine-lysine) by liquid chromatography-mass spectrometry.

detailed description

The invention is further illustrated by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are generally in accordance with the conventional conditions in the art or according to the conditions recommended by the manufacturer; the raw materials, reagents and the like used are commercially available from conventional markets, unless otherwise specified. The raw materials and reagents obtained. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of the invention as claimed.

Example 1

This embodiment provides an improved cellulose having the following structural formula:

As shown in Fig. 2, the improved cellulose provided by the present invention is prepared by the following preparation method:

(1) Prepare a certain size of laboratory filter paper (Whatman 50, its structural form is shown in Figure 1), put it into a stainless steel container, and add a sufficient amount of dimethylformamide (DMF) to the stainless steel container (washing step). Gently shake for 1 hour, then pour in the solvent (repeated 3 times); then add enough dichloromethane (DCM) to a stainless steel container, gently shake for 10 minutes, then pour the solvent (repeated 3 times), air dry;

(2) The air-dried paper cellulose film is placed in a stainless steel container, and a pyridine solution of p-toluenesulfonyl chloride (380 g of toluenesulfonyl chloride dissolved in 1 L of pyridine) is added to the stainless steel container, and gently shaken at room temperature for 15 minutes. Add enough DMF (washing step) to the stainless steel container, gently shake for 20 minutes, then pour the solvent (repeated 3 times); add enough DCM stainless steel container, shake gently for 10 minutes, then pour the solvent (repeated 3 (times), air-dried paper cellulose;

(3) placing the air-dried paper cellulose membrane in a stainless steel container, adding a sufficient amount of 4,7,10 trioxo-1,13-tridecanediamine (TTDDA), and gently shaking at room temperature overnight; Add enough dimethylformamide (DMF) to the stainless steel vessel (washing step), shake gently for 1 hour, then pour in the solvent (repeated 3 times); add enough dichloromethane (DCM) to the stainless steel container. The modified cellulose was obtained by gently shaking for 10 minutes and then pouring the solvent (repeated 3 times).

Example 2

The improved cellulose provided by the present invention is prepared by the following preparation method:

(1) Prepare a certain size of laboratory filter paper (Whatman 50), put it into a stainless steel container, add a sufficient amount of dimethylformamide (DMF) to the stainless steel container (washing step), and gently shake for 1 hour. Then pour in the solvent (repeated 3 times); then add enough dichloromethane (DCM) to the stainless steel container, gently shake for 10 minutes, then pour the solvent (repeated 3 times), air dry;

(2) The air-dried paper cellulose film is placed in a stainless steel container, and a sufficient amount of a solution of p-toluenesulfonyl chloride in pyridine (350 g of tosyl chloride dissolved in 1 L of pyridine) is added to the stainless steel container, and gently shaken at room temperature for 15 minutes. Add enough DMF (washing step) to the stainless steel container, gently shake for 20 minutes, then pour the solvent (repeated 3 times); add enough DCM stainless steel container, shake gently for 10 minutes, then pour the solvent (repeated 3 (times), air-dried paper cellulose;

(3) placing the air-dried paper cellulose film in a stainless steel container, adding a sufficient amount of Bis(3-aminopropoxy)ethane, and gently shaking at room temperature overnight; Add enough dimethylformamide (DMF) to the stainless steel vessel (washing step), shake gently for 1 hour, then pour in the solvent (repeated 3 times); add enough dichloromethane (DCM) to the stainless steel container. The modified cellulose was obtained by gently shaking for 10 minutes and then pouring the solvent (repeated 3 times).

Example 3

(2) Place the air-dried paper cellulose film in a stainless steel container, add a sufficient amount of p-toluenesulfonyl chloride in pyridine solution (400 gram of toluenesulfonyl chloride dissolved in 1 L of pyridine), and gently shake for 15 minutes at room temperature. Add enough DMF (washing step) to the stainless steel container, gently shake for 20 minutes, then pour the solvent (repeated 3 times); add enough DCM stainless steel container, shake gently for 10 minutes, then pour the solvent (repeated 3 (times), air-dried paper cellulose;

(3) Place the air-dried paper cellulose film in a stainless steel container and add a sufficient amount of Hepta (ethylene glycol) bis (3-aminopropyl) at room temperature. Shake gently overnight; add sufficient amount of dimethylformamide (DMF) to the stainless steel vessel (washing step), gently shake for 1 hour, then pour in the solvent (repeated 3 times); add enough dichloromethane (DCM) The modified cellulose was obtained by gently shaking for 10 minutes in a stainless steel container and then pouring the solvent (repeated 3 times).

Example 4

This embodiment provides a modified cellulose modified by Rink Amide linker, and its structural formula is as follows:

As shown in Fig. 2, the modified cellulose provided in this embodiment is prepared by the following preparation method:

(3) placing the air-dried paper cellulose membrane in a stainless steel container, adding a sufficient amount of 4,7,10 trioxo-1,13-tridecanediamine (TTDDA), and gently shaking at room temperature overnight; Add enough dimethylformamide (DMF) to the stainless steel vessel (washing step), shake gently for 1 hour, then pour in the solvent (repeated 3 times); add enough dichloromethane (DCM) to the stainless steel container. Gently shake for 10 minutes, then pour in the solvent (repeated 3 times) and air dry;

(4) The paper cellulose which was air-dried in step (3) was placed in a stainless steel container, and 0.7 mol of Rink Amide linker was added (the structural formula was:

), 0.7 mol of hydroxybenzotriazole (HOBt), 0.7 mol of N,N'-diisopropylcarbimine DIC, and solvent DMF, vortexed at 70 ° C for 15 minutes, and then sequentially used DMF, ethanol (2 times) and Wash with DCM and air dry.

Example 5

(3) placing the air-dried paper cellulose film in a stainless steel container, adding a sufficient amount of Bis(3-aminopropoxy)ethane, and gently shaking at room temperature overnight; Add enough dimethylformamide (DMF) to the stainless steel vessel (washing step), shake gently for 1 hour, then pour in the solvent (repeated 3 times); add enough dichloromethane (DCM) to the stainless steel container. Gently shake for 10 minutes, then pour in the solvent (repeated 3 times) and air dry;

), 0.7 mol of hydroxybenzotriazole (HOBt), 0.7 mol of N,N'-diisopropylcarbimine DIC and solvent DMF, gently shake at 50 ° C for 40 minutes, and then sequentially use DMF, ethanol (2 times) ) Wash with DCM and air dry.

Example 6

(3) Place the air-dried paper cellulose film in a stainless steel container and add a sufficient amount of Hepta (ethylene glycol) bis (3-aminopropyl) at room temperature. Shake gently overnight; add sufficient amount of dimethylformamide (DMF) to the stainless steel vessel (washing step), gently shake for 1 hour, then pour in the solvent (repeated 3 times); add enough dichloromethane (DCM) ) into a stainless steel container, gently shake for 10 minutes, then pour the solvent (repeated 3 times), air dry;

), 0.7 mol of hydroxybenzotriazole (HOBt), 0.7 mol of N,N'-diisopropylcarbimine DIC, and solvent DMF, vortexed at 80 ° C for 10 minutes, and then sequentially used DMF, ethanol (2 times) and Wash with DCM and air dry.

Example 7

This embodiment provides a modified cellulose modified by Wang linker, which has the following structural formula:

(4) Put the paper cellulose dried in step (3) in a stainless steel container, and add 0.1 mol of Wang linker, 0.11 mmol of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline. EEDQ and 0.1 mol of hexamethylphosphoric triamide HMPA and NMP (N-methylpyrrolidone) solvent were shaken at 20 ° C for 14 hours, then washed successively with DMF, ethanol (2 times) and DCM, and air-dried.

Example 8

Example 9

(2) The air-dried paper cellulose film is placed in a stainless steel container, and a sufficient amount of a solution of p-toluenesulfonyl chloride in pyridine (400 g of toluenesulfonyl chloride dissolved in 1 L of pyridine) is added to the stainless steel container, and gently shaken at room temperature for 15 minutes. Add enough DMF (washing step) to the stainless steel container, gently shake for 20 minutes, then pour the solvent (repeated 3 times); add enough DCM stainless steel container, shake gently for 10 minutes, then pour the solvent (repeated 3 (times), air-dried paper cellulose;

Application example 1

Taking the modified cellulose provided in Example 1 as an example, it was used as a matrix for solid phase polypeptide synthesis, and the performance was tested by ultraviolet light absorption and Western blotting (Western Blotting).

Synthesis was carried out from the C-terminus to the N-terminus using a polypeptide synthesizer. The schematic process of the synthesis is shown in Figure 3. The synthesized polypeptide structure is shown in Figure 4A. Bioactive peptide 6 repeat histidine (HHHHHH) is a positive control for synthesis and detection. The synthesized polypeptide is tyrosine-serine-valine-threonine-serine-valine-serine (YSPTSPS) and repeat structure (YSPTSPSYSPTSPS) and wherein the 2,5,7-serine is phosphatidylserine ( s) Substituted sequence polypeptide. Figure 4B shows the results of the absorption of the synthesized polypeptide in ultraviolet light, all of which are positive results; since the synthetic polypeptides are similar in structure, they absorb in the ultraviolet, and there is no significant difference. Figure 4C shows the results of Western Blotting. The biological activity is different due to the amino acid sequence of the polypeptide. In the Western Blotting assay, there is a clear difference in the position of the modified amino acid (phosphatidylserine); The difference between the 2,5 phosphatidylserine polypeptide (YsPTsPS) and its repeat structure (YsPTsPSYsPTsPS) was also shown. As can be seen from the figure, the modified cellulose provided in the present embodiment can be used as a matrix for solid polypeptide synthesis, and the polypeptide is successfully synthesized, and the yield and yield of the synthesis are suitable for the activity scanning of the polypeptide.

Application example 2

The properties of the modified cellulose were tested by taking the modified cellulose modified by Rink Amide linker provided in Example 4 as an example.

The solid peptide synthesis was carried out using the modified cellulose modified by Rink Amide linker provided in Example 4, and the synthesized polypeptide was tested. The steps are: synthesis by automatic synthesizer, cutting protection group and modified cellulose modified by Rink Amide linker, mass spectrometry synthesis results.

Synthesis was carried out from the C-terminus to the N-terminus using a polypeptide synthesizer. The schematic process of the synthesis is shown in Figure 5. Take the synthetic peptide proline-valine-valine-lysine-lysine (Ver-Ver-Ver-Ver-Lys) as an example: first wet and clean Rink with DMF and ethanol. Amide linker modified modified cellulose, Fmoc protection with pyridine solution, the first step is to add the C-terminal amino acid - lysine (Lys), then repeat the deprotection and synthesis steps to synthesize the desired peptide; finally use pyridine Go to protect. The Rink Amide linker modified modified cellulose-synthesized polypeptide (such as Ver-Ver-Ver-Ver-Lys) was cleaved, and each SPOT polypeptide was cleaved with 100 ul of 90% trifluoroacetic acid, 3% triisopropylsilane. , 2% water and 5% dichloromethane, gently shake for 2 hours.

The synthesized peptide (valine-valine-valine-valine-lysine) was detected by liquid chromatography-mass spectrometry and its molecular weight was 541.3, which was shown in liquid chromatography-mass spectrometry (Fig. 6). The main compound has a proton ion peak of 542.4 at a retention time of 8.64 minutes and is a synthetic target polypeptide. It can be seen from Fig. 6 that the polypeptide (proline-valine-valine-valine-lysine) has been successfully synthesized, and the modified cellulose has good performance and can be used as a matrix for solid polypeptide synthesis. Good reactivity and synthetic purity.

Application Example 3

The properties of the modified cellulose were tested by using the Wang linker modified modified cellulose provided in Example 7.

The solid peptide synthesis was carried out using the modified cellulose modified by Wang linker provided in Example 7, and the synthesized polypeptide was tested. The steps are: automatic synthesizer synthesis, cutting protection group and Wang linker modified modified cellulose on the cut, mass spectrometry synthesis results.

Synthesis was carried out from the C-terminus to the N-terminus using a polypeptide synthesizer. The schematic process of the synthesis is shown in Figure 7. Take the synthetic peptide proline-valine-valine-lysine-lysine (Ver-Ver-Ver-Ver-Lys) as an example: first wet and clean Wang with DMF and ethanol. The linker modified modified cellulose is protected by Fmoc with a pyridine solution. The first step is to add the C-terminal amino acid, lysine (Lys), and then repeat the deprotection and synthesis steps to synthesize the desired polypeptide; protection. The modified linker-modified polypeptide (such as Ver-Ver-Ver-Ver-Lys) modified by Wang linker is cleaved, and each SPOT polypeptide is cleaved with 100 ul of 90% trifluoroacetic acid and 3% triisopropylsilane. 2% water and 5% dichloromethane, gently shake for 2 hours.

The synthesized polypeptide (valine-valine-valine-valine-lysine) was detected by liquid chromatography-mass spectrometry and its molecular weight was 542.3, which was shown in liquid chromatography-mass spectrometry (Fig. 8). The main compound has a proton ion peak of 543.39 at a retention time of 10.18 minutes, which is a synthetic target polypeptide. As can be seen from Fig. 8, the polypeptide (proline-valine-valine-valine-lysine) was successfully synthesized, and the modified cellulose has excellent performance and can be used as a matrix for solid polypeptide synthesis. Has good reactivity and synthetic purity.

Claims

A novel modified cellulose characterized in that the structural formula of the modified cellulose is as shown in formula (I), formula (II) or formula (III):

Wherein n is 2 to 7; the modified cellulose is obtained by modifying the cellulose.
The improved cellulose of claim 1 wherein said cellulose is paper cellulose.
The improved cellulose according to claim 2, wherein the cellulose is a filter paper.
The improved cellulose of claim 1 wherein n is 3.
The method for preparing modified cellulose according to any one of claims 1 to 4, wherein the method comprises the steps of:

S1: mixing a solution of cellulose and p-toluenesulfonyl chloride in pyridine, shaking, washing and air drying;

S2: adding long chain compounds
After shaking until the reaction is complete, wash,

Air-dried, the modified cellulose of formula (I);

S3: adding the Ring Amide resin and the activator to the modified cellulose of the formula (I), and then washing the modified cellulose to obtain the modified cellulose of the formula (II);

S4: adding the Wang linker and the activator to the modified cellulose of the formula (I), followed by washing and then washing to obtain the modified cellulose of the formula (III).
The method for producing modified cellulose according to claim 5, wherein the concentration of p-toluenesulfonyl chloride in the pyridine solution of the p-toluenesulfonyl chloride in S1 is from 350 to 400 g/L.
The method for producing modified cellulose according to claim 6, wherein the mass concentration of p-toluenesulfonyl chloride in the pyridine solution of the p-toluenesulfonyl chloride of S1 is 380 g/L.
The method for preparing modified cellulose according to claim 5, wherein the activator in S3 is HOBt hydroxybenzotriazole and DIC N, N'-diisopropylcarbimine, or HOSu N- Hydroxysuccinimide and DIC N,N'-diisopropylcarbinimine; the reaction temperature is 50-80 ° C, the reaction time is 10-40 min; the activator in S4 is HMPA hexamethylphosphorus Acetyltriamine and EEDQ 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; the reaction temperature is 18-25 ° C, and the reaction time is 12-16 h.
A method of preparing modified cellulose according to claim 5, further comprising the step of subjecting the cellulose to an anhydrous treatment before the step S1.
Use of the improved cellulose according to any one of claims 1 to 4 as a substrate for the synthesis of solid polypeptides.